The present focus of this project is to understand how DNA supercoiling is brought about by DNA gyrase and related enzymes, and to study the effects of DNA supercoiling on bacterial gene expression. These are some recent results: The extremely slow binding of the ATP analog (beta, gamma-imido) ATP to DNA gyrase is greatly accelerated in the presence of ATP. Thus paradoxically ATP can help the analog to block ATP binding sites and inhibit the enzyme. The effect of DNA supercoiling on bacterial transcription has been further studied. In a promoter that is induced by DNA relaxation, the structure of the first transcribed region turns out to be important; when the DNA is supercoiled, many transcripts terminate prematurely, while full-length transcripts are formed on relaxed DNA. Experiments on the reverse DNA gyrase of thermophilic bacteria have shown that, unlike all other topoisomerases tested, this enzyme can only change supercoiling in one direction (toward positive supercoiling). Reverse gyrase is ATP-dependent, but can function with a remarkably low concentration of ATP.